The Src homology 2 (SH2) domain of the mammalian adaptor protein Crk-II contains a proline-rich insert, predicted to lie within an extended DE loop, which is dispensable for phosphopeptide binding. Using the yeast two-hybrid system, this region of the Crk-II SH2 domain was found to interact with a subset of SH3 domains, notably the Abl SH3 domain. Furthermore, this proline-rich insert was found to modify the efficiency with which Crk-II was phosphorylated by the p140(c-abl) tyrosine kinase. In vitro, the interaction of full-length non-phosphorylated Crk-II with a glutathione S-transferase-Abl SH3 domain fusion protein was very weak. However, phosphorylation of Crk-II on Tyr-221 which induces an intramolecular association with the SH2 domain, or addition of a phosphopeptide corresponding to the Crk-II Tyr-221 phosphorylation site, stimulated association of Crk-II with the Abl SH3 domain. NMR spectroscopic analysis showed that binding of the Tyr-221 phosphopeptide to the Crk SH2 domain induced a chemical shift change in Val-71, located in the proline-rich insert, indicative of a change in the structure of the proline-rich loop in response of Crk SH2-pTyr-221 interaction. These results suggest that the proline-rich insert in the Crk SH2 domain constitutes an SH3 domain-binding site that can be regulated by binding of a phosphopeptide ligand to the Crk SH2 domain.